The present invention relates to analytical instruments, and more particularly to Electrospray and Liquid chromatography (LC)/Mass spectrometry (MS) interfaces and the miniaturization of these interfaces.
In understanding physical phenomenon, there is often a desire to obtain the molecular weight and structure of a given compound. Mass spectrometry (MS) is a technique that addresses this desire. Using this technique, molecules are weighed by ionizing the molecule itself and measuring the response of the ion, or ions, as they traverse an electric and magnetic field. In the early development of Mass spectrometry, the mechanisms of ionization were electron ionization, chemical ionization and photo-ionization. These methods of ionization were useful for low molecular weight molecules. However, relatively large molecules could not be subjected to Mass spectrometric analysis using these modes of ionization. This left a large gap in the ability to understand, for example, certain biological phenomena. Clearly there existed a need to develop techniques applicable to large molecular weight molecules.
Ionization techniques were improved, such as field desorption, thermospray and Electrospray. These soft ionization techniques produce intact molecular ions that originate from high molecular weight molecules. As a result of this development, applications of Mass spectrometry to biological investigation have become increasingly more frequent. The precise application will often determine which soft ionization technique is to be employed, however, Electrospray is an often used soft ionization technique for producing molecular weight data on large biomolecules.
A liquid flowing through a capillary jet or orifice may be converted into a spray of small charged droplets (on the order of 1 xcexcm in diameter) by applying a strong electric field to the liquid as it emerges from the tip of a capillary. For a sufficiently high applied field, the electrostatic stress imposed by the field and the surface-induced electrical charge is sufficient to overcome the surface tension forces on the liquid. Breaking apart into a large number of small charged droplets is a way for the liquid to disperse the charge and reach a lower total energy state. This process of forming a spray is commonly referred to as Electrospray.
Presently, apparatus are available for forming an Electrospray of a sample solution such as a liquid stream effluent from a Liquid chromatography separation step, and subsequently analyzing the Electrospray with a Mass analyzer such as a quadrupole Mass spectrometer, an ion trap, a time-of-flight Mass spectrometer, or a magnetic sector Mass spectrometer, or the like. Interposed between the apparatus for forming the Electrospray and the analytical apparatus are means for desolvating the droplets forming the Electrospray so as to form a stream of intact ions which are to be analyzed. In a liquid chromatograph, a stream of solvent, containing a mixture of chemical species in solution, is passed at elevated pressure through a chromatographic column. The column is designed so that it separates the mixture by differential retention on the column into its component species. The different species then emerge from the column as distinct bands in the solvent stream, separated in time. Coupling the output of a liquid chromatograph to a Mass spectrometer via an Electrospray interface gives the analyst a powerful tool since it can provide molecular weight and structural information about the separated species as they emerge from the liquid chromatograph.
A variety of Electrospray interfaces are known in the industry, such as those described in U.S. Pat. No. 5,756,994 to Bajic, U.S. Pat. No. 4,977,320 to Chowdhury et al., U.S. Pat. No. 5,015,845 to Allen et al., U.S. Pat. No. 4,999,493 to Allen et al., U.S. Pat. No. 4,531,056 to Labowsky et al., U.S. Pat. No. 5,304,798 to Tomany et al, U.S. Pat. No. 4,861,988 to Henion et al, U.S. Pat. No. 4,209,696 to Fite and UK Pat. No. 1,246,709 to Hazelby et al.
These techniques primarily involve electrostatic nebulization with, or without, pneumatic, thermal, or ultrasonic assistance in forming droplets of a liquid stream containing analyte. These droplets may be generated in a heated or unheated gas stream. The gas serves to desolvate the droplets. The droplets subsequently shrink as they desolvate, resulting in the formation of atmospheric ions of the analyte. These ions and gas and solvent vapors are then sampled into a region of lower pressure where they can be analyzed using Mass spectrometry.
Additionally, interfaces using time modulated Electrosprays are described in U.S. Pat. No. 5,436,446 to Jarrell et al. and U.S. Pat. No. 5,306910 to Jarrell et al. Time modulated Electrosprays form discontinuous bursts of spray that facilitate spray manipulation.
The performance of any interface may be characterized by the efficiency with which sample molecules are ionized, and then the efficiency with which these formed ions are transported into the analyzing means. These prior methods are deficient, however, in that they lack substantial means for the concentration of ions prior to the transfer of these ions into a region of lower pressure entering into the region of low pressure.
The utility of using these soft ionization techniques for coupling the output of a separation to Mass analyzers has been amply demonstrated in the last decade. There is now an additional need to reduce the cost and size of analytical equipment based on these techniques. Miniaturization impacts the utility of these techniques in many ways, improving the cost, reducing the size, improving the reliability and reducing the expense of reagents and concomitantly, the cost of used reagent disposal.
Recently progress has been made in the miniaturization of separation technology. Capillary zone electrophoresis and Liquid chromatography have been implemented on micromachined substrates. Similarly, progress has been made in the miniaturization of Mass analyzers. For example, Ramsey and coworkers have developed a Micro Ion Trap Mass Spectrometer as reported in abstract No. ThPB027 at the 1999 American Society for Mass Spectrometry Conference in Dallas, Tex. In order to enable a fully miniaturized LC/MS or CZE/MS or CEC/MS based system, there is the need for means to miniaturize the interface between the separation techniques and the Mass analyzer. Karger (U.S. Pat. No. 5,872,010) has demonstrated means to miniaturize the generation of the means for forming a conventional Electrospray. While interesting, this has little utility if the goal is to produce a fully miniaturized analytical instrument.
Hence there is a need for methods and apparatus to miniaturize a complete interface.
The present invention provides improved methods and apparatus for coupling the output from liquid phase separation techniques to Mass spectrometry, in particular, Liquid chromatography/Mass spectrometry (LC/MS). Further, these methods and apparatus are capable of being miniaturized.
According to an illustrative embodiment, the charged droplets (from which ions are formed) are produced in a controlled manner, one at a time or xe2x80x9con demandxe2x80x9d. The droplets are formed by mechanical means. A voltage is used to charge the droplets, but unlike conventional Electrospray, the voltage supplied is insufficient to directly cause the formation of an Electrospray. This is in contrast to prior art techniques where the droplets are produced in large numbers with very little control. Controlling the means of droplet generation, increases the efficiency with which subsequent necessary steps can occur. This increased efficiency, enables a further embodiment in which the structures necessary for a complete interface can be reduced in scale sufficiently to enable their construction using various micromachining techniques.
The subsequent steps of desolvation, ion generation, ion concentration, introduction of said formed ions into a lower pressure (vacuum) region that embody a complete interface can be distributed in space or in time. Prior art has focussed on processes that are distributed in space as these are more easily optimized for ion streams that are essentially continuous. The controlled production of single droplets, however, makes feasible the effective distribution of these processes in time. Additionally, this controlled production of droplets and hence ions enables effective means for concentrating these droplets and/or ions prior to the transfer of these ions into a region of lower pressure.
According to a further embodiment of the invention, additional flows of gas are provided in a nebulization region to effectively remove undesired solvent vapor and concentrate desirable analyte containing droplets and/or analyte ions. A porous structure is used to effectively separate associated gas flow streams. Ions are concentrated using electrodes which are appropriately electrically biased and driven with DC and RF voltages so as to concentrate the ions in a laminar gas stream. Additionally, tapered wedges or skimmers are positioned so as to peel away the portion of the gas stream that contains, for example, a majority of the ions.
A significant advantage that is derived from the current invention is an increased efficiency with respect to ion formation and transportation.